Treatment of ischemia

ABSTRACT

A system, including methods and compositions, for treatment of ischemia.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application claims priority under U.S. and international law(including but not limited to the Paris Convention and 35 U.S.C. §119(e)) to U.S. Provisional Patent Application Ser. No. 60/611,241 (U.S.60/611,241), filed Sep. 16, 2004, which is incorporated herein byreference in its entirety for all purposes.

GOVERNMENT LICENSE RIGHTS

This invention was made with U.S. Government support under grant R21NS42799 from the National Institutes of Health. The U.S. Government thusmay have certain license rights in this invention.

BACKGROUND

Strokes may be caused by a disruption of blood flow to the brain, forexample, due to a clot or a leak in a blood vessel that supplies thebrain with blood. This disruption of blood flow deprives brain tissue ofoxygen, often resulting in localized death of brain tissue (focalinfarction) and thus permanent damage to the brain.

Changes in the ion flux into neurons may lead to the cell death producedby stroke. Accordingly, various ion channels may be candidates formediating this altered ion flux, thus confounding the search for asuitable therapeutic target.

SUMMARY

The present teachings provide a system, including methods andcompositions, for treatment of ischemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of a flowchart illustrating an exemplary method oftreating ischemia, in accordance with aspects of the present teachings.

FIG. 2 is a view of a flowchart illustrating an exemplary method ofidentifying drugs for treating ischemia, in accordance with aspects ofthe present teachings.

FIG. 3 is a series of graphs presenting exemplary data related to theelectrophysiology and pharmacology of acid sensing ion channel (ASIC)proteins in cultured mouse cortical neurons, in accordance with aspectsof the present teachings.

FIG. 4 is an additional series of graphs presenting exemplary datarelated to the electrophysiology and pharmacology of ASIC proteins incultured mouse cortical neurons, in accordance with aspects of thepresent teachings.

FIG. 5 is a set of graphs and traces presenting exemplary data showingthat modeled ischemia may enhance activity of ASIC proteins, inaccordance with aspects of the present teachings.

FIGS. 6 and 7 are a set of graphs and traces presenting exemplary datashowing that ASIC proteins in cortical neurons may be Ca²⁺ permeable,and that Ca²⁺ permeability may be ASIC1a dependent, in accordance withaspects of the present teachings.

FIG. 8 is a series of graphs presenting exemplary data showing that acidincubation may induce glutamate receptor-independent neuronal injurythat is protected by ASIC blockade, in accordance with aspects of thepresent teachings.

FIG. 9 is a series of graphs presenting exemplary data showing thatASIC1a may be involved in acid-induced injury in vitro, in accordancewith aspects of the present teachings.

FIG. 10 is a series of graphs with data showing neuroprotection in brainischemia in vivo by ASIC1a blockade and by ASIC1 gene knockout, inaccordance with aspects of the present teachings.

FIG. 11 is a graph plotting exemplary data for the percentage ofischemic damage produced by stroke in an animal model system as afunction of the time and type of treatment, in accordance with aspectsof the present teachings.

FIG. 12 is a view of the primary amino acid sequence of an exemplarycystine knot peptide, PcTx1, with various exemplary peptide featuresshown, in accordance with aspects of the present teachings.

FIG. 13 is a comparative view of the cystine knot peptide of FIG. 12aligned with various exemplary deletion derivatives of the peptide, inaccordance with aspects of the present teachings.

FIG. 14 is an exemplary graph plotting the amplitude of calcium currentmeasured in cells as a function of the ASIC family member(s) expressedin the cells, in accordance with aspects of the present teachings.

FIG. 15 is a graph presenting exemplary data related to the efficacy ofnasally administered PcTx venom in reducing ischemic injury in an animalmodel system, in accordance with aspects of the present teachings.

DETAILED DESCRIPTION

The present teachings provide a system, including methods andcompositions, for treatment of ischemia. The methods may includeapproaches for reducing injury resulting from ischemia and/or foridentifying drugs for ischemia treatment. The methods selectively mayinhibit one or more members of the acid sensing ion channel (ASIC)family, to provide a targeted therapy for ischemia treatment.

FIG. 1 shows a flowchart 20 with exemplary steps 22, 24 that may beperformed in a method of treating ischemia. The steps may be performedany suitable number of times and in any suitable combination. In themethod, an ischemic subject (or subjects) may be selected for treatment,indicated at 22. An ASIC-selective inhibitor then may be administered tothe ischemic subject(s), indicated at 24. Administration of theinhibitor to the ischemic subject may be in a therapeutically effectamount, to reduce ischemia-induced injury to the subject, for example,reducing the amount of brain damage resulting from a stroke.

A potential explanation for the efficacy of the ischemia treatment ofFIG. 1 may be offered by the data of the present teachings (e.g., seeExample 1). In particular, the damaging effects of ischemia may not beequal to acidosis, that is, acidification of tissue/cells via ischemiamay not be sufficient to produce ischemia-induced injury. Instead,ischemia-induced injury may be caused, in many cases, by calcium fluxinto cells mediated by a member(s) of the ASIC family, particularlyASIC1a. Accordingly, selective inhibition of the channel activity ofASIC la may reduce this harmful calcium flux, thereby reducingischemia-induced injury.

FIG. 2 shows a flowchart 30 with exemplary steps 32, 34 that may beperformed in a method of identifying drugs for treatment of ischemia.The steps may be performed any suitable number of times and in anysuitable combination. In the method, one or more ASIC-selectiveinhibitors may be obtained, indicated at 32. The inhibitors then may betested on an ischemic subject for an effect on ischemia-induced injury,indicated at 34.

The methods of the present teachings may provide one or more advantagesover other methods of ischemia treatment. These advantages may include(1) less ischemia-induced injury, (2) fewer side effects of treatment(e.g., due to selection of a more specific therapeutic target), and/or(3) a longer time window for effective treatment, among others.

Further aspects of the present teachings are described in the followingsections, including (I) ischemia, (II) ischemic subjects and subjectselection, (III) ASIC inhibitors, (IV) administration of inhibitors, (V)identification of drugs, and (VI) examples.

I. Ischemia

The system of the present teachings is directed to treatment of anysuitable ischemia. Ischemia, as used herein, is a reduced blood flow toan organ(s) and/or tissue(s). The reduced blood flow may be caused byany suitable mechanism including a partial or complete blockage (anobstruction), a narrowing (a constriction), and/or a leak/rupture, amongothers, of one or more blood vessels that supply blood to the organ(s)and/or tissue(s). Accordingly, ischemia may be created by thrombosis, anembolism, atherosclerosis, hypertension, hemorrhage, an aneurysm,surgery, trauma, medication, and/or the like. The reduced blood flowthus may be chronic, transient, acute, sporadic, and/or the like.

Any suitable organ or tissue may experience a reduced blood flow in theischemia being treated. Exemplary organs and/or tissues may include thebrain, arteries, the heart, intestines, the eye (e.g., the optic nerve),etc. Ischemia-induced injury (i.e., disease and/or damage) produced byvarious ischemias may include ischemic myelopathy, ischemic opticneuropathy, ischemic colitis, coronary heart disease, and/or cardiacheart disease (e.g., angina, heart attack, etc.), among others.Ischemia-induced injury thus may damage and/or kill cells and/or tissue,for example, producing necrotic (infarcted) tissue, inflammation, and/ortissue remodeling, among others, at affected sites within the body.Treatment according to aspects of the present teachings may reduce theincidence, extent, and/or severity of this injury.

The system of the present teachings may provide treatment of stroke.Stroke, as used herein, is brain ischemia produced by a reduced bloodsupply to a part (or all) of the brain. Symptoms produced by stroke maybe sudden (such as loss of consciousness) or may have a gradual onsetover hours or days. Furthermore, the stroke may be a major ischemicattack (a full stroke) or a more minor, transient ischemic attack, amongothers. Symptoms produced by stroke may include, for example,hemiparesis, hemiplegia, one-sided numbness, one-sided weakness,one-sided paralysis, temporary limb weakness, limb tingling, confusion,trouble speaking, trouble understanding speech, trouble seeing in one orboth eyes, dim vision, loss of vision, trouble walking, dizziness, atendency to fall, loss of coordination, sudden severe headache, noisybreathing, and/or loss of consciousness. Alternatively, or in addition,the symptoms may be detectable more readily or only via tests and/orinstruments, for example, an ischemia blood test (e.g., to test foraltered albumin, particular protein isoforms, damaged proteins, etc.),an electrocardiogram, an electroencephalogram, an exercise stress test,and/or the like.

II. Ischemic Subjects and Subject Selection

The system of the present teachings may provide treatment of ischemicsubjects to reduce ischemic injury to the subjects. An ischemic subject,as used herein, is any person (a human subject) or animal (an animalsubject) that has ischemia, an ischemia-related condition, a history ofischemia, and/or a significant chance of developing ischemia aftertreatment begins and during a time period in which the treatment isstill effective.

The ischemic subject may be an animal. The term “animal,” as usedherein, refers to any animal that is not human. Exemplary animals thatmay be suitable include any animal with a bloodstream, such as rodents(mice, rats, etc.), dogs, cats, birds, sheep, goats, non-human primates,etc. The animal may be treated for its own sake, e.g., for veterinarypurposes (such as treatment of a pet). Alternatively, the animal mayprovide an animal model of ischemia, to facilitate testing drugcandidates for human use, such as to determine the candidates' potency,window of effectiveness, side effects, etc. Further aspects of testingperformed with animal model systems are described below in Section V.

An ischemia-related condition may be any consequence of ischemia. Theconsequence may be substantially concurrent with the onset ischemia(e.g., a direct effect of the ischemia) and/or may occur substantiallyafter ischemia onset and/or even after the ischemia is over (e.g., anindirect, downstream effect of the ischemia, such reperfusion of tissuewhen ischemia ends). Exemplary ischemia-related conditions may includeany combination of the symptoms (and/or conditions) listed above inSection I. Alternatively, or in addition, the symptoms may include localand/or systemic acidosis (pH decrease), hypoxia (oxygen decrease), freeradical generation, and/or the like.

Ischemic subjects for treatment may be selected by any suitablecriteria. Exemplary criteria may include any detectable symptoms ofischemia, a history of ischemia, an event that increases the risk of (orinduces) ischemia (such as a surgical procedure, trauma, administrationof a medication, etc.), and/or the like. A history of ischemia mayinvolve one or more prior ischemic episodes. In some examples, a subjectselected for treatment may have had an onset of ischemia that occurredat least about one, two, or three hours before treatment begins, or aplurality of ischemic episodes (such as transient ischemic attacks) thatoccurred less than about one day, twelve hours, or six hours prior toinitiation of treatment.

III. ASIC Inhibitors

Inhibitors of ASIC family members, as used herein, are substances thatreduce (partially, substantially, or completely block) the activity orone or more members of the ASIC family, that is, ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3, and ASIC4, among others. In some examples, the inhibitorsmay reduce the channel activity of one or more members, such as theability of the members to flux ions (e.g., sodium, calcium, and/orpotassium ions, among others) through cell membranes (into and/or out ofcells). The substances may be compounds (small molecules of less thanabout 10 kDa, peptides, nucleic acids, lipids, etc.), complexes of twoor more compounds, and/or mixtures, among others. Furthermore, thesubstances may inhibit ASIC family members by any suitable mechanismincluding competitive, noncompetitive, uncompetitive, and/or mixedinhibition, among others.

The inhibitor may be an ASIC1a inhibitor that inhibits acid sensing ionchannel 1a (ASIC1a). ASIC1a, as used herein, refers to an ASIC1a proteinor channel from any species. For example, an exemplary human ASIC1aprotein/channel is described in Waldmann, R., et al. 1997, Nature 386,pp. 173-177, which is incorporated herein by reference.

The expression “ASIC1a inhibitor” may refer to a product which, withinthe scope of sound pharmacological judgment, is potentially or actuallypharmaceutically useful as an inhibitor of ASIC1a, and includesreference to substances which comprise a pharmaceutically active speciesand are described, promoted, or authorized as an ASIC1a inhibitor.

An ASIC1a inhibitor may be selective within the ASIC family. Selectiveinhibition of ASIC1a, as used herein, is inhibition that issubstantially stronger on ASIC1a than on another ASIC family member(s)when compared (for example, in cultured cells) after exposure of each tothe same (sub-maximal) concentration(s) of an inhibitor. The inhibitormay inhibit ASIC1a selectively relative to at least one other ASICfamily member (ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC 4, etc.) and/orselectively relative to every other ASIC family member. The strength ofinhibition for a selective inhibitor may be described by an inhibitorconcentration at which inhibition occurs (e.g., an IC₅₀ (inhibitorconcentration that produces 50% of maximal inhibition) or a K_(i) value(inhibition constant or dissociation constant)) relative to differentASIC family members. An ASIC1a-selective inhibitor may inhibit ASIC1aactivity at a concentration that is at least about two-, four-, orten-fold lower (one-half, one-fourth, or one-tenth the concentration orlower) than for inhibition of at least one other or of every other ASICfamily member. Accordingly, an ASIC1a-selective inhibitor may have anIC₅₀ and/or K_(i) for ASIC1a inhibition that is at least about two-,four-, or ten-fold lower (one-half, one-fourth, or one-tenth or less)than for inhibition of at least one other ASIC family member and/or forinhibition of every other ASIC family member.

An ASIC1a-selective inhibitor, in addition to being selective, also maybe specific for ASIC1a. ASIC1a-specific inhibition, as used herein, isinhibition that is substantially exclusive to ASIC1a relative to everyother ASIC family member. An ASIC1a-specific inhibitor may inhibitASIC1a at an inhibitor concentration that is at least about twenty-foldlower (5% of the concentration or less) than for inhibition of everyother ASIC family member. Accordingly, an ASIC1a-specific inhibitor mayhave an IC₅₀ and/or K_(i) for ASIC1a relative to every other member ofthe ASIC family that is at least about twenty-fold lower (five percentor less), such that, for example, inhibition of other ASIC familymembers is at least substantially (or completely) undetectable.

Any suitable ASIC inhibitor or combination of inhibitors may be used.For example, a subject may be treated with an ASIC1a-selective inhibitorand a nonselective ASIC inhibitor, or with an ASIC1a-selective inhibitorand an inhibitor to a non-ASIC channel protein, such as a non-ASICcalcium channel. In some examples, a subject may be treated with anASIC1a-selective inhibitor and an inhibitor of NMDA receptors, such as aglutamate antagonist.

The inhibitor may be or include a peptide. The peptide may have anysuitable number of amino acid subunits, generally at least about ten andless than about one-thousand subunits. In some examples, the peptide mayhave a cystine knot motif. A cystine knot, as used herein, generallycomprises an arrangement of six or more cysteines. A peptide with thesecysteines may create a “knot” including (1) a ring formed by twodisulfide bonds and their connecting backbone segments, and (2) a thirddisulfide bond that threads through the ring. In some examples, thepeptide may be a conotoxin from an arachnid and/or cone snail species.For example, the peptide may be PcTx1 (psalmotoxin 1), a toxin from atarantula (Psalmopoeus cambridgei (Pc)).

In some examples, the peptide may be structurally related to PcTx1, suchthat the peptide and PcTx1 differ by at least one deletion, insertion,and/or substitution of one or more amino acids. For example, the peptidemay have at least about 25% or at least about 50% sequence identity,and/or at least about 25% or at least about 50% sequence similarity withPcTx1 (see below). Further aspects of peptides that may be suitable asinhibitors are described below in Example 3.

Methods of alignment of amino acid sequences for comparison andgeneration of identity and similarity scores are well known in the art.Exemplary alignment methods that may be suitable include (Best Fit) ofSmith and Waterman, a homology alignment algorithm (GAP) of Needlemanand Wunsch, a similarity method (Tfasta and Fasta) of Pearson andLipman, and/or the like. Computer algorithms of these and otherapproaches that may be suitable include, but are not limited to:CLUSTAL, GAP, BESTFIT, BLASTP, FASTA, and TFASTA.

As used herein, “sequence identity” or “identity” in the context of twopeptides relates to the percentage of residues in the correspondingpeptide sequences that are the same when aligned for maximumcorrespondence. In some examples, peptide residue positions that are notidentical may differ by conservative amino acid substitutions, whereamino acid residues are substituted for other amino acid residues withsimilar chemical properties (e.g. charge or hydrophobicity) andtherefore are expected to produce a smaller (or no) effect on thefunctional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards, to give a “similarity” of the sequences, whichcorrects for the conservative nature of the substitutions. For example,each conservative substitution may be scored as a partial rather than afull mismatch, thereby correcting the percentage sequence identity toprovide a similarity score. The scoring of conservative substitutions toobtain similarity scores is well known in the art and may be calculatedby any suitable approach, for example, according to the algorithm ofMeyers and Miller, Computer Applic. Biol Sci., 4: 11-17 (1988), e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

IV. Administration of Inhibitors

Administration (or administering), as used herein, includes any route ofsubject exposure to an inhibitor, under any suitable conditions, and atany suitable time(s). Administration may be self-administration oradministration by another, such as a health-care practitioner (e.g., adoctor, a nurse, etc.). Administration may be by injection (e.g.,intravenous, intramuscular, subcutaneous, intracerebral, epidural,and/or intrathecal, among others), ingestion (e.g., using a capsule,lozenge, a fluid composition, etc.), inhalation (e.g., an aerosol (lessthan about 10 microns average droplet diameter) inhaled nasally and/ororally), absorption through the skin (e.g., with a skin patch) and/ormucosally (e.g., through oral, nasal, and/or pulmonary mucosa, amongothers), and/or the like. Mucosal administration may be achieved, forexample, using a spray (such as a nasal spray), an aerosol that isinhaled), and/or the like. A spray may be a surface spray (droplets onaverage greater than about 50 microns in diameter) and/or a space spray(droplets on average about 10-50 microns in diameter). In some examples,ischemia may produce an alteration of the blood-brain barrier of anischemic subject, thus increasing the efficiency with which an inhibitorthat is introduced (e.g., by injection and/or absorption) into thebloodstream of a subject can reach the brain. Administration may beperformed once or a plurality of times, and at any suitable timerelative to ischemia diagnosis, to provide treatment. Accordingly,administration may be performed before ischemia has been detected (e.g.,prophylactically,) after a minor ischemic episode, during chronicischemia, after a full stroke, and/or the like.

A therapeutically effective amount of an inhibitor may be administered.A therapeutically effective amount of an inhibitor, as used herein, isany amount of the inhibitor that, when administered to subjects,reduces, in a significant number of the subjects, the degree, incidence,and/or extent of ischemia-induced injury in the subjects. Accordingly, atherapeutically effective amount may be determined, for example, inclinical studies in which various amounts of the inhibitor areadministered to test subjects (and, generally, compared to a controlgroup of subjects).

The inhibitor may be administered in any suitable form and in anysuitable composition to subjects. In some examples, the inhibitor may beconfigured as a pharmaceutically acceptable salt. The composition may beformulated to include, for example, a fluid carrier/solvent (a vehicle),a preservative, one or more excipients, a coloring agent, a flavoringagent, a salt(s), an anti-foaming agent, and/or the like. The inhibitormay be present at a concentration in the vehicle that provides atherapeutically effective amount of the inhibitor for treatment ofischemia when administered to an ischemic subject.

V. Identification of Drugs

Additional ASIC inhibitors may be identified for use as drugs to treatischemia. Identification may include (A) obtaining one or more ASICinhibitors, and (B) testing the ASIC inhibitors on ischemic subjects.

A. Obtaining ASIC Inhibitors

One or more ASIC inhibitors, particularly ASIC1a inhibitors as describedabove, may be obtained. The inhibitors may be obtained by any suitableapproach, such by screening a set of candidate inhibitors (e.g., alibrary of two or more compounds) and/or by rationale design, amongothers.

Screening may involve any suitable assay system that measuresinteraction between ASIC proteins and the set of candidate inhibitors.Exemplary assay systems may include assays performed biochemically(e.g., binding assays), with cells grown in culture (“cultured cells”),and/or with organisms, among others.

A cell-based assay system may measure an effect, if any, of eachcandidate inhibitor on ion flux in the cells, generally acid-sensitiveion flux. In some examples, the ion flux may be a flux of calcium and/orsodium, among others. The assay system may use cells expressing an ASICfamily member, such as ASIC1a, or two or more distinct sets of cellsexpressing two or more distinct ASIC family members, such as ASIC1a andanother ASIC family member(s), to determine the selectivity of eachinhibitor for these family members. The cells may express each ASICfamily member endogenously or through introduction of foreign nucleicacid. In some examples, the assay system may measure ion fluxelectrophysiologically (such as by patch clamp), using an ion-sensitiveor membrane potential-sensitive dye (e.g., a calcium sensitive dye suchas Fura-2), or via a gene-based reporter system that is sensitive tochanges in membrane potential and/or intracellular ion (e.g., calcium)concentrations, among others. The assay system may be used to testcandidate inhibitors for selective and/or specific inhibition of ASICfamily members, particularly ASIC1a.

B. Testing ASIC Inhibitors on Subjects

One or more ASIC inhibitors may be administered to an ischemicsubject(s) to test the efficacy of the inhibitors for treatment ofischemia. The ischemic subjects may be people or animals. In someexamples, the ischemic subjects may provide an animal model system ofischemia and/or stroke. Exemplary animal model systems include rodents(mice and/or rats, among others) with ischemia induced experimentally.The ischemia may be induced mechanically (e.g., surgically) and/or byadministration of a drug, among others. In some examples, the ischemiamay be induced by occlusion of a blood vessel, such as by constrictionof a mid-cerebral artery.

VI. EXAMPLES

The following examples describes selected aspects and embodiments of thepresent teachings, particularly data describing in vitro and in vivoeffects of ASIC inhibition, and exemplary cystine knot peptides for useas inhibitors. These examples are intended for the purposes ofillustration and should not be construed to limit the scope of thepresent teachings.

Example 1 Neuroprotection in Ischemia Blocking Calcium-PermeableAcid-Sensing Ion Channels

This example describes experiments showing a role of ASIC1a in mediatingischemic injury and the ability ASIC1a inhibitors to reduce ischemicinjury; see FIGS. 2-10.

A. Overview

Ca²⁺ toxicity may play a central role in ischemic brain injury. Themechanism by which toxic Ca²⁺ loading of cells occurs in the ischemicbrain has become less clear as multiple human trials of glutamateantagonists have failed to show effective neuroprotection in stroke.Acidosis may be a common feature of ischemia and may play a criticalrole in brain injury; however, the mechanism(s) remains ill defined.Here, we show that acidosis may activate Ca²⁺-permeable acid-sensing ionchannels (ASICs), which may induce glutamate receptor-independent,Ca²⁺-dependent, neuronal injury inhibited by ASIC blockers. Cellslacking endogenous ASICs may be resistant to acid injury, whiletransfection of Ca²⁺-permeable ASIC1a may establish sensitivity. Infocal ischemia, intracerebroventricular injection of ASIC1a blockers orknockout of the ASIC1a gene may protect the brain from ischemic injuryand may do so more potently than glutamate antagonism. Thus, acidosismay injure the brain via membrane receptor-based mechanisms withresultant toxicity of [Ca²⁺] (intracellular calcium), disclosing newpotential therapeutic targets for stroke.

B. Introduction

Intracellular Ca²⁺ overload may be important for neuronal injuryassociated with neuropathological syndromes, including brain ischemia(Choi 1995 and Choi 1988a). Excessive Ca²⁺ in the cell may activate acascade of cytotoxic events leading to activation of enzymes that breakdown proteins, lipids, and nucleic acids. NMDA receptors, which may bethe most important excitatory neurotransmitter receptors in the centralnervous system (McLennan 1983 and Dingledine et al. 1999), have longbeen considered the main target responsible for Ca²⁺ overload in theischemic brain (Simon et al. 1984; Rothman and Olney 1986; Choi 1988band Meldrum 1995). However, recent clinical efforts to prevent braininjury through the therapeutic use of NMDA receptor antagonists havebeen disappointing (Lee et al. 1999 and Wahlgren and Ahmed 2004).Although multiple factors, including difficulty in early initiation oftreatment, may have contributed to trial failures, glutamatereceptor-independent Ca²⁺ toxicity also or alternatively may beresponsible for ischemic brain injury.

The normal brain may require complete oxidation of glucose to fulfillits energy requirements. During ischemia, oxygen depletion may force thebrain to switch to anaerobic glycolysis. Accumulation of lactic acid asa byproduct of glycolysis and protons produced by ATP hydrolysis maycause pH to fall in the ischemic brain (Rehncrona 1985 and Siesjo et al.1996). Consequently, tissue pH typically falls to 6.5-6.0 duringischemia under normoglycemic conditions and may fall below 6.0 duringsevere ischemia or under hyperglycemic conditions (Nedergaard et al.1991; Rehncrona 1985 and Siesjo et al. 1996). Nearly all in vivo studiesindicate that acidosis aggravates ischemic brain injury (Tombaugh andSapolsky 1993 and Siesjo et al. 1996). However, the mechanisms of thisprocess remain unclear, although a host of possibilities has beensuggested (Siesjo et al. 1996; McDonald et al. 1998; Swanson et al. 1995and Ying et al. 1999).

Acid-sensing ion channels (ASICs), a newly described class ofligand-gated channels (Waldmann et al. 1997a and Krishtal 2003), havebeen shown to be expressed throughout neurons of mammalian central andperipheral nervous systems (Waldmann et al. 1997a; Waldmann et al. 1999;Waldmann and Lazdunski 1998; Krishtal 2003; Alvarez de la Rosa et al.2002 and Alvarez de la Rosa et al. 2003). These channels are members ofthe degenerin/epithelial sodium channel (Deg/ENaC) superfamily (Benosand Stanton 1999; Bianchi and Driscoll 2002 and Krishtal 2003).Pertinent to ischemia, ASICs also may flux Ca²⁺ (Waldmann et al. 1997a;Chu et al. 2002 and Yermolaieva et al. 2004).

To date, six ASIC subunits have been cloned. Four of these subunits mayform functional homomultimeric channels that are activated by acidic pHto conduct a sodium-selective, amiloride-sensitive, cation current. ThepH of half-maximal activation (pH_(0.5)) of these channels differs:ASIC1a, pH_(0.5)=6.2 (Waldmann et al., 1997a); ASIC1β (also termedASIC1b), a splice variant of ASIC1a with a unique N-terminal,pH_(0.5)=5.9 (Chen et al., 1998); ASIC2a, pH_(0.5)=4.4 (Waldmann et al.,1999); and ASIC3, pH_(0.5)=6.5 (Waldmann et al., 1997b). Neither ASIC2bnor ASIC4 can form functional homomeric channel (Akopian et al. 2000;Grunder et al. 2000 and Lingueglia et al. 1997), but ASIC2b has beenshown to associate with other subunits and modulate their activity(Lingueglia et al., 1997). In addition to Na⁺ permeability, homomericASIC1a may flux Ca²⁺ (Waldmann et al. 1997a; Chu et al. 2002 andYermolaieva et al. 2004). Although the exact subunit composition ofASICs in native neurons has not been determined, both ASIC1a and ASIC2asubunits have been shown to be abundant in the brain (Price et al. 1996;Bassilana et al. 1997; Wemmie et al. 2002 and Alvarez de la Rosa et al.2003).

Detailed functions of ASICs in both peripheral and central nervoussystems remain to be determined. In peripheral sensory neurons, ASICshave been implicated in mechanosensation (Price et al. 2000 and Price etal. 2001) and perception of pain during tissue acidosis (Bevan and Yeats1991; Krishtal and Pidoplichko 1981; Ugawa et al. 2002; Sluka et al.2003 and Chen et al. 2002), particularly in ischemic myocardium whereASICs likely transduce anginal pain (Benson et al., 1999). The presenceof ASICs in the brain, which lacks nociceptors, suggests that thesechannels may have functions beyond nociception. Indeed, recent studieshave indicated that ASIC1a may be involved in synaptic plasticity,learning/memory, and fear conditioning (Wemmie et al. 2002 and Wemmie etal. 2003). Here, using a combination of patch-clamp recording, Ca²⁺imaging, receptor subunit transfection, in vitro cell toxicity assays,and in vivo ischemia models combined with gene knockout, we demonstrateactivation of Ca²⁺-permeable ASIC1a as largely responsible forglutamate-independent, acidosis-mediated, and ischemic brain injury.

C. Results

1. Acidosis Activates ASICs in Mouse Cortical Neurons

FIGS. 3 and 4 shows exemplary data related to the electrophysiology andpharmacology of ASICs in cultured mouse cortical neurons. FIGS. 3A and3B are graphs illustrating the pH dependence of ASIC currents activatedby a pH drop from 7.4 to the pH values indicated. Dose-response curveswere fit to the Hill equation with an average pH_(0.5) of 6.18±0.06(n=10). FIGS. 3C and 3D are graphs illustrating the current-voltagerelationship of ASICs (n=5). The amplitudes of ASIC current at variousvoltages were normalized to that recorded at −60 mV. FIGS. 4A and 4B aregraphs illustrating a dose-dependent blockade of ASIC currents byamiloride. IC₅₀=16.4±4.1 μM, N=8. FIGS. 4C and 4D are graphsillustrating a blockade of ASIC currents by PcTX venom. **p<0.01.

We first recorded ASIC currents in cultured mouse cortical neurons, apreparation commonly used for cell toxicity studies (Koh and Choi 1987and Sattler et al. 1999); see FIG. 3. At a holding potential of −60 mV,a rapid reduction of extracellular pH (pH_(e)) to below 7.0 evoked largetransient inward currents with a small steady-state component in themajority of neurons (FIG. 3A). The amplitude of inward current increasedin a sigmoidal fashion as pH_(e) decreased, yielding a pH_(0.5) of6.18±0.06 (n=10, FIG. 3B). A linear I-V relationship and a reversalclose to the Na⁺ equilibrium potential were obtained (n=6, FIGS. 3C and3D). These data demonstrate that lowering pH_(e) may activate typicalASICs in mouse cortical neurons.

We then tested the effect of amiloride, a nonspecific blocker of ASICs(Waldmann et al., 1997a), on the acid-activated currents; see FIG. 4.Similar to previous studies, mainly in sensory neurons (Waldmann et al.1997a; Benson et al. 1999; Chen et al. 1998 and Varming 1999), amiloridedose-dependently blocked ASIC currents in cortical neurons with an IC₅₀of 16.4±4.1 μM (n=8, FIGS. 4A and 4B). Psalmotoxin 1 (or PcTX1) fromvenom of the tarantula Psalmopoeus cambridgei (PcTX venom) may be aspecific ASIC1a blocker (Escoubas et al., 2000). Our studies show that,at a protein concentration of 25 ng/mL, PcTX venom itself may block thecurrent mediated by homomeric ASIC1a expressed in COS-7 cells by ˜70%(n=4, see Supplemental Figure S1 athttp://www.cell.com/cgi/content/full/118/6/687/DC1, which isincorporated herein by reference). However, it does not affect currentsmediated by heteromeric ASIC1a/2a, homomeric ASIC2a, or ASIC3 channelsat 500 ng/mL (n=4-6). In addition, at 500 ng/mL, PcTX venom does notaffect the currents through known voltage- and ligand-gated channels,further indicating its specificity for homomeric ASIC1a (n=4-5,Supplemental Figure S2, and Supplemental Data (at the website citedabove), which are incorporated herein by reference).

We then tested the effect of PcTX venom on acid-activated current incortical neurons. At 100 ng/mL, PcTX venom reversibly blocked the peakamplitude of ASIC current by 47%±7% (n=15, FIGS. 4C and 4D), indicatingsignificant contributions of homomeric ASIC1a to total acid-activatedcurrents. Increasing PcTX concentration did not induce further reductionin the amplitude of ASIC current in the majority of cortical neurons(n=8, data not shown), indicating coexistence of PcTX-insensitive ASICs(e.g., heteromeric ASIC1a/2a) in these neurons.

2. ASIC Response Is Potentiated by Modeled Ischemia

FIG. 5 shows exemplary data indicating that modeled ischemia may enhanceactivity of ASICs. FIG. 5A is a series of exemplary traces showing anincrease in amplitude and a decrease in desensitization of ASIC currentsfollowing 1 hr OGD. FIG. 5B is a graph of summary data illustrating anincrease of ASIC current amplitude in OGD neurons. N=40 and 44, *p<0.05.FIG. 5C is a series of exemplary traces and summary data showingdecreased ASIC current desensitization in OGD neurons. N=6, **p<0.01.FIG. 5D is a pair of exemplary traces showing lack of acid-activatedcurrent at pH 6.0 in ASIC1^(−/−) neurons, in control condition, andfollowing 1 hr OGD (n=12 and 13).

Since acidosis may be a central feature of brain ischemia, we determinedwhether ASICs may be activated in ischemic conditions and whetherischemia may modify the properties of these channels; see FIG. 5. Werecorded ASIC currents in neurons following I hr oxygen glucosedeprivation (OGD), a common model of in vitro ischemia (Goldberg andChoi, 1993). One set of cultures was washed three times withglucose-free extracellular fluid (ECF) and subjected to OGD, whilecontrol cultures were subjected to washes with glucose containing ECFand incubation in a conventional cell culture incubator. OGD wasterminated after 1 hr by replacing glucose-free ECF with Neurobasalmedium and incubating cultures in the conventional incubator. ASICcurrent was then recorded 1 hr following the OGD when there was nomorphological alteration of neurons. OGD treatment induced a moderateincrease of the amplitude of ASIC currents (1520±138 pA in controlgroup, N=44; 1886±185 pA in neurons following 1 hr OGD, N=40, p<0.05,FIGS. 5A and 5B). More importantly, OGD induced a dramatic decrease inASIC desensitization as demonstrated by an increase in time constant ofthe current decay (814.7±58.9 ms in control neurons, N=6; 1928.9±315.7ms in neurons following OGD, N=6, p<0.01, FIGS. 5A and 5C). In corticalneurons cultured from ASIC1^(−/−) mice, reduction of pH from 7.4 to 6.0did not activate any inward current (n=52), similar to a previous studyin hippocampal neurons (Wemmie et al., 2002). In these neurons, 1 hr OGDdid not activate or potentiate acid-induced responses (FIG. 5D, n=12 and13).

3. Acidosis Induces Glutamate-Independent Ca2+ Entry via ASIC1a

FIGS. 6 and 7 show exemplary data suggesting that ASICs in CorticalNeurons may be Ca²⁺ permeable, and that Ca²⁺ permeability may be ASIC1adependent. FIG. 6A shows exemplary traces obtained with Na⁺-free ECFcontaining 10 mM Ca²⁺ as the only charge carrier. Inward currents wererecorded at pH 6.0. The average reversal potential is ˜−17 mV aftercorrection of liquid junction potential (n=5). FIG. 6B showsrepresentative traces and summary data illustrating blockade ofCa²⁺-mediated current by amiloride and PcTX venom. The peak amplitude ofCa²⁺-mediated current decreased to 26%±2% of control value by 100 μMamiloride (n=6, p<0.01) and to 22%±0.9% by 100 ng/mL PcTX venom (n=5,p<0.01). FIG. 7A shows exemplary 340/380 nm ratios as a function of pH,illustrating an increase of [Ca²⁺]_(i) by pH drop to 6.0. Neurons werebathed in normal ECF containing 1.3 mM CaCl₂ with blockers forvoltage-gated Ca²⁺ channels (5 μM nimodipine and 1 μM ω-conotoxin MVIIC)and glutamate receptors (10 μM MK801 and 20 μM CNQX). The inset of FIG.7A shows exemplary inhibition of acid-induced increase of [Ca²⁺]_(i) by100 μM amiloride. FIG. 7B shows exemplary summary data illustratinginhibition of acid-induced increase of [Ca²⁺]_(i) by amiloride and PcTXvenom. N=6-8, **p<0.01 compared with pH 6.0 group. FIG. 7C showsexemplary 340/380 nm ratios as a function of pH and NMDApresence/absence, demonstrating a lack of acid-induced increase of[Ca²⁺]_(i) in ASIC1^(−/−) neurons; neurons had a normal response to NMDA(n=8). FIG. 7D shows exemplary traces illustrating a lack ofacid-activated current at pH 6.0 in ASIC1^(−/−) neurons.

Using a standard ion-substitution protocol (Jia et al., 1996) and theFura-2 fluorescent Ca2+-imaging technique (Chu et al., 2002), wedetermined whether ASICs in cortical neurons are Ca²⁺ permeable; seeFIGS. 6 and 7. With bath solutions containing 10 mM Ca²⁺ (Na⁺ andK⁺-free) as the only charge carrier and at a holding potential of −60mV, we recorded inward currents larger than 50 pA in 15 out of 18neurons, indicating significant Ca²⁺ permeability of ASICs in themajority of cortical neurons (FIG. 6A). Consistent with activation ofhomomeric ASIC1a channels, currents carried by 10 mM Ca²⁺ were largelyblocked by both the nonspecific ASIC blocker amiloride and theASIC1a-specific blocker PcTX venom (FIG. 6B). The peak amplitude ofCa²⁺-mediated current was decreased to 26%±2% of control by 100 μMamiloride (n=6, p<0.01) and to 22%±0.9% by 100 ng/mL PcTX venom (n=5,p<0.01). Ca²⁺ imaging, in the presence of blockers of other major Ca²⁺entry pathways (MK801 10 μM and CNQX 20 μM for glutamate receptors;nimodipine 5 μM and ω-conotoxin MVIIC 1 μM for voltage-gated Ca²⁺channels), demonstrated that 18 out of 20 neurons responded to a pH dropwith detectable increases in the concentration of intracellular Ca²⁺([Ca²⁺]_(i)) (FIG. 7A). In general, [Ca²⁺]_(i) remains elevated duringprolonged perfusion of low pH solutions. In some cells, the [Ca²⁺]_(i)increase lasted even longer than the duration of acid perfusion (FIG.7A). Long-lasting Ca²⁺ responses suggest that ASIC response in intactneurons may be less desensitized than in whole-cell recordings or thatCa²⁺ entry through ASICs may induce subsequent Ca²⁺ release fromintracellular stores. Preincubation of neurons with 1 μM thapsigarginpartially inhibited the sustained component of Ca²⁺ increase, suggestingthat Ca²⁺ release from intracellular stores may also contribute toacid-induced intracellular Ca²⁺ accumulation (n=6, data not shown).Similar to the current carried by Ca²⁺ ions (FIG. 6B), both peak andsustained increases in [Ca²⁺]_(i) were largely inhibited by amilorideand PcTX venom (FIGS. 7A and 7B, n=6-8), consistent with involvement ofhomomeric ASIC1a in acid-induced [Ca²⁺]_(i) increase. Knockout of theASICI gene eliminated the acid-induced [Ca²⁺]_(i) increase in allneurons without affecting NMDA receptor-mediated Ca²⁺ response (FIG. 7C,n=8). Patch-clamp recordings demonstrated lack of acid-activatedcurrents at pH 6.0 in 52 out of 52 of the ASIC1^(−/−) neurons,consistent with absence of ASIC1a subunits. Lowering pH to 5.0 or 4.0,however, activated detectable current in 24 out of 52 ASIC1^(−/−)neurons, indicating the presence of ASIC2a subunits in these neurons(FIG. 7D). Further electrophysiological studies demonstrated thatASIC1^(−/−) neurons have normal responses for various voltage-gatedchannels and NMDA, GABA receptor-gated channels (data not shown).

4. ASIC Blockade Protects Acidosis-Induced, Glutamate-IndependentNeuronal Injury

FIG. 8 shows exemplary data suggesting that acid incubation may induceglutamate receptor-independent neuronal injury protected by ASICblockade. FIGS. 8A and 8B show graphs presenting exemplary data fortime-dependent LDH release induced by 1 hr (FIG. 8A) or 24 hr incubation(FIG. 8B) of cortical neurons in pH 7.4 (solid bars) or 6.0 ECF (openbars). N=20-25 wells, *p<0.05, and **p<0.01, compared to pH 7.4 group atthe same time points. (Acid-induced neuronal injury with fluoresceindiacetate (FDA) also was analyzed by staining of cell bodies of aliveneurons and propidium iodide (PI) staining of nuclei of dead neurons.)FIG. 8C shows a graph illustrating inhibition of acid-induced LDHrelease by 100 μM amiloride or 100 ng/mL PcTX venom (n=20-27, *p<0.05,and **p<0.01). MK801, CNQX, and nimodipine were present in ECF for allexperiments (FIGS. 8A-C).

Acid-induced injury was studied on neurons grown on 24-well platesincubated in either pH 7.4 or 6.0 ECF containing MK801, CNQX, andnimodipine; see FIG. 8. Cell injury was assayed by the measurement oflactate dehydrogenase (LDH) release (Koh and Choi, 1987) at various timepoints (FIGS. 8A and 8B) and by fluorescent staining of alive/deadcells. Compared to neurons treated at pH 7.4, 1 hr acid incubation (pH6.0) induced a time-dependent increase in LDH release (FIG. 8A). After24 hr, 45.7%±5.4% of maximal LDH release was induced (n=25 wells).Continuous treatment at pH 6.0 induced greater cell injury (FIG. 8B,n=20). Consistent with the LDH assay, alive/dead staining withfluorescein diacetate (FDA, blue) and propidium iodide (PI, red) showedsimilar increases in cell death by 1 hr acid treatment (see SupplementalFigure S3 (on the web site cited above), which is incorporated herein byreference). One hour incubation with pH 6.5 ECF also induced significantbut less LDH release than with pH 6.0 ECF (n=8 wells).

To determine whether activation of ASICs is involved in acid-inducedglutamate receptor-independent neuronal injury, we tested the effect ofamiloride and PcTX venom on acid-induced LDH release. Addition of either100 μM amiloride or 100 ng/mL PcTX venom 10 min before and during the 1hr acid incubation significantly reduced LDH release (FIG. 8C). At 24hr, LDH release was decreased from 45.3%±3.8% to 31.1%±2.5% by amilorideand to 27.9%±2.6% by PcTX venom (n=20-27, p<0.01). Addition of amilorideor PcTX venom in pH 7.4 ECF for 1 hr did not affect baseline LDHrelease, although prolonged incubation (e.g., 5 hr) with amiloride aloneincreased LDH release (n=8).

5. Activation of Homomeric ASIC1a May be Responsible forAcidosis-Induced Injury

FIG. 9 is a series of graphs presenting exemplary data indicating thatASIC1a may be involved in acid-induced injury in vitro. FIG. 9A showsexemplary data illustrating inhibition of acid-induced LDH release byreducing [Ca²⁺]_(e) (n=11-12, **p<0.01 compared with pH 6.0, 1.3 Ca²⁺).FIG. 9B shows exemplary data illustrating acid incubation inducedincrease of LDH release in ASIC1a-transfected but not nontransfectedCOS-7 cells (n=8-20). Amiloride (100 μM) inhibited acid-induced LDHrelease in ASIC1a-transfected cells. *p<0.05 for 7.4 versus 6.0 and 6.0versus 6.0+amiloride. FIG. 9C shows exemplary data illustrating a lackof acid-induced injury and protection by amiloride and PcTX venom inASIC1^(−/−) neurons (n=8 in each group, p>0.05). FIG. 9D shows exemplarydata illustrating acid-induced increase of LDH release in culturedcortical neurons under OGD (n=5). LDH release induced by combined 1 hrOGD/acidosis was not inhibited by trolox and L-NAME (n=8-11). OGD didnot potentiate acid-induced LDH release in ASIC1^(−/−) neurons. **p<0.01for pH 7.4 versus pH 6.0 and *p<0.05 for pH 6.0 versus 6.0+PcTX venom.MK801, CNQX, and nimodipine were present in ECF for all experiments(FIG. 9A-D).

To determine whether Ca²⁺ entry plays a role in acid-induced injury, wetreated neurons with pH 6.0 ECF in the presence of normal or reduced[Ca²⁺]_(e); see FIG. 9. Reducing Ca²⁺ from 1.3 to 0.2 mM inhibitedacid-induced LDH release (from 40.0%±4.1% to 21.9%±2.5%), as did ASIC1ablockade with PcTX venom (n=11-12, p<0.01; FIG. 9A). Ca²⁺-free solutionwas not tested, as a complete removal of [Ca²⁺]_(e) may activate largeinward currents through a Ca²⁺-sensing cation channel, which mayotherwise complicate data interpretation (Xiong et al., 1997).Inhibition of acid injury by both amiloride and PcTX, nonspecific andspecific ASIC1a blockers, and by reducing [Ca²⁺]_(e) suggests thatactivation of Ca²⁺-permeable ASIC1a may be involved in acid-inducedneuronal injury.

To provide additional evidence that activation of ASIC1a is involved inacid injury, we studied acid injury of nontransfected and ASIC1atransfected COS-7 cells, a cell line commonly used for expression ofASICs due to its lack of endogenous channels (Chen et al. 1998; Immkeand McCleskey 2001 and Escoubas et al. 2000). Following confluence(36-48 hr after plating), cells were treated with either pH 7.4 or 6.0ECF for 1 hr. LDH release was measured 24 hr after acid incubation.Treatment of nontransfected COS-7 cells with pH 6.0 ECF did not induceincreased LDH release when compared with pH 7.4-treated cells(10.3%±0.8% for pH 7.4, and 9.4%±0.7% for pH 6.0, N=19 and 20 wells;p>0.05, FIG. 9B). However, in COS-7 cells stably transfected withASIC1a, 1 hr incubation at pH 6.0 significantly increased LDH releasefrom 15.5%±2.4% to 24.0%±2.9% (n=8 wells, p<0.05). Addition of amiloride(100 μM) inhibited acid-induced LDH release in these cells (FIG. 9B).

We also studied acid injury of CHO cells transiently transfected withcDNAs encoding GFP alone or GFP plus ASIC1a. After the transfection(24-36 hr), cells were incubated with acidic solution (pH 6.0) for 1 hr,and cell injury was assayed 24 hr following the acid incubation. Asshown in Supplemental Figure S4 (at the website cited above), which isincorporated herein by reference, 1 hr acid incubation largely reducedsurviving GFP-positive cells in GFP/ASIC1a group but not in the grouptransfected with GFP alone (n=3 dishes in each group).

To further demonstrate an involvement of ASIC1a in acidosis-inducedneuronal injury, we performed cell toxicity experiments on corticalneurons cultured from ASIC^(+/+) and ASIC1^(−/−) mice (Wemmie et al.,2002). Again, 1 hr acid incubation of ASIC^(+/+) neurons at 6.0 inducedsubstantial LDH release that was reduced by amiloride and PcTX venom(n=8-12). One hour acid treatment of ASIC1^(−/−) neurons, however, didnot induce significant increase in LDH release at 24 hr (13.8%±0.9% forpH 7.4 and 14.2%±1.3% for pH 6.0, N=8, p>0.05), indicating resistance ofthese neurons to acid injury (FIG. 9C). In addition, knockout of theASIC1 gene also eliminated the effect of amiloride and PcTX venom onacid-induced LDH release (FIG. 9C, n=8 each), further suggesting thatthe inhibition of acid-induced injury of cortical neurons by amilorideand PcTX venom (FIG. 8C) was due to blockade of ASIC1 subunits. Incontrast to acid incubation, 1 hr treatment of ASIC1^(−/−) neurons with1 mM NMDA+10 μM glycine (in Mg²⁺-free [pH 7.4] ECF) induced 84.8%±1.4%of maximal LDH release at 24 hr (n=4, FIG. 9C), indicating normalresponse to other cell injury processes.

6. Modeled Ischemia Enhances Acidosis-Induced Glutamate-IndependentNeuronal Injury Via ASICs

As the magnitude of ASIC currents may be potentiated by cellular andneurochemical components of brain ischemia—cell swelling, arachidonicacid, and lactate (Allen and Attwell 2002 and Immke and McCleskey2001)—and, more importantly, the desensitization of ASIC currents may bereduced dramatically by modeled ischemia (see FIGS. 5A and 5C), weexpected that activation of ASICs in ischemic conditions should producegreater neuronal injury. To test this hypothesis, we subjected neuronsto 1 hr acid treatment under oxygen and glucose deprivation (OGD).MK801, CNQX, and nimodipine were added to all solutions to inhibitvoltage-gated Ca²⁺ channels and glutamate receptor-mediated cell injuryassociated with OGD (Kaku et al., 1991). One hour incubation with pH 7.4ECF under OGD conditions induced only 27.1%±3.5% of maximal LDH releaseat 24 hr (n=5, FIG. 9D). This finding is in agreement with a previousreport that 1 hr OGD does not induce substantial cell injury with theblockade of glutamate receptors and voltage-gated Ca²⁺ channels (Aartset al., 2003). However, 1 hr OGD, combined with acidosis (pH 6.0),induced 73.9%±4.3% of maximal LDH release (n=5, FIG. 9D, p<0.01),significantly larger than acid-induced LDH release in the absence of OGD(see FIG. 8A, p<0.05). Addition of the ASIC1a blocker PcTX venom (100ng/mL) significantly reduced acid/OGD-induced LDH release to 44.3%±5.3%(n=5, p<0.05, FIG. 9D).

We also performed the same experiment with cultured neurons from theASIC1^(−/−) mice. Unlike in ASICI containing neurons, however, 1 hrtreatment with combined OGD and acid only slightly increased LDH releasein ASIC1^(−/−) neurons (from 26.1%±2.7% to 30.4%±3.5%, N=10-12, FIG.9D). This finding suggests that potentiation of acid-induced injury byOGD may be due largely to OGD potentiation of ASIC1-mediated toxicity.

Aarts et al. (2003) have recently studied ischemia molded by prolongedOGD (2 hr) but without acidosis. In this model system, they demonstratedactivation of a Ca²⁺-permeable nonselective cation conductance activatedby reactive oxygen/nitrogen species resulting in glutamatereceptor-independent neuronal injury. The prolonged OGD-induced cellinjury modeled by Aarts et al. may be reduced dramatically by agentseither scavenging free radicals directly (e.g., trolox) or reducing theproduction of free radicals (e.g., L-NAME) (Aarts et al., 2003). Todetermine whether combined short duration OGD and acidosis inducedneuronal injury may involve a similar mechanism, we tested the effect oftrolox and L-NAME on OGD/acid-induced LDH release. As shown in FIG. 9D,neither trolox (500 μM) nor L-NAME (300 μM) had significant effect oncombined 1 hr OGD/acidosis-induced neuronal injury (n=8-11). Additionalexperiments demonstrated that the ASIC blockers amiloride and PcTX venomhad no effect on the conductance of TRPM7 channels reported to beresponsible for prolonged OGD-induced neuronal injury by Aarts et al.(2003) (Supplemental Figure S5 (see website listed above), which isincorporated herein by reference). Together, these findings stronglysuggest that activation of ASICs but not TRPM7 channels may be largelyresponsible for combined 1 hr OGD/acidosis-induced neuronal injury inour studies.

7. Activation of ASIC1a in Ischemic Brain Injury In Vivo

FIG. 10 shows data illustrating neuroprotection by ASICI blockade andASICI gene knockout in brain ischemia in vivo. FIG. 10A shows a graph ofexemplary data obtained from TTC-stained brain sections illustrating thestained volume (“infarct volume”) in brains from aCSF (n=7), amiloride(n=11), or PcTX venom (n=5) injected rats. *p<0.05 and **p<0.01 comparedwith aCSF injected group. FIG. 10B shows a graph of exemplary dataillustrating reduction in infarct volume in brains from ASIC1^(−/−) mice(n=6 for each group). *p<0.05 and **p<0.01 compared with +/+ group. FIG.10C shows a graph of exemplary data illustrating reduction in infarctvolume in brains from mice i.p. injected with 10 mg/kg memantine (Mem)or i.p. injection of memantine accompanied by i.c.v. injection of PcTXvenom (500 ng/mL). **p<0.01 compared with aCSF injection and betweenmemantine and memantine plus PcTX venom (n=5 in each group). FIG. 10Dshows a graph of exemplary data illustrating reduction in infarct volumein brains from either ASIC1^(+/+) (wt) or ASIC1^(−/−) mice i.p. injectedwith memantine (n=5 in each group). *p<0.05, and **p<0.01.

To provide evidence that activation of ASIC1a may be involved inischemic brain injury in vivo, we first tested the protective effect ofamiloride and PcTX venom in a rat model of transient focal ischemia(Longa et al., 1989). Ischemia (100 min) was induced by transient middlecerebral artery occlusion (MCAO). A total of 6 μl artificial CSF (aCSF)alone, aCSF-containing amiloride (1 mM), or PcTX venom (500 ng/mL) wasinjected intracerebroventricularly 30 min before and after the ischemia.Based on the study by Westergaard (1969), the volume for cerebralventricular and spinal cord fluid for 4-week-old rats is estimated to be˜60 μl. Assuming that the infused amiloride and PcTX were uniformlydistributed in the CSF, we may expect a concentration of 100 μM foramiloride and ˜50 ng/mL for PcTX, which is a concentration foundeffective in our cell culture experiments. Infarct volume was determinedby TTC staining (Bederson et al., 1986) at 24 hr following ischemia.Ischemia (100 min) produced an infarct volume of 329.5±25.6 mm³ inaCSF-injected rats (n=7) but only 229.7±41.1 mm³ in amiloride-injected(n=11, p<0.05) and 130.4±55.0 mm³ (˜60% reduction) in PcTXvenom-injected rats (n=5, p<0.01) (FIG. 10A).

We next used ASIC1^(−/−) mice to further demonstrate the involvement ofASIC1a in ischemic brain injury in vivo. Male ASIC1^(+/+), ASIC1^(+/−),and ASIC1^(−/−) mice (˜25 g, with congenic C57B16 background) weresubjected to 60 min MCAO as previously described (Stenzel-Poore et al.,2003). Consistent with the protection by pharmacological blockade ofASIC1a (above), −/− mice displayed significantly smaller (˜61%reduction) infarct volumes (32.9±4.7 mm³, N=6) as compared to +/+ mice(84.6±10.6 mm³, N=6, p<0.01).+/− mice also showed reduced infarct volume(56.9±6.7 mm³, N=6, p<0.05) (FIG. 10B).

We then determined whether blockade of ASIC1a channels or knockout ofthe ASIC1 gene could provide additional protection in vivo in thesetting of glutamate receptor blockade. We selected the uncompetitiveNMDA receptor antagonist memantine, as it has been recently used insuccessful clinical trials (Tariot et al., 2004). Memantine (10 mg/kg)was injected intraperitoneally (i.p.) into C57B16 mice immediatelyfollowing 60 min MCAO and accompanied by intracerebroventricularinjection (i.c.v.) of a total volume of 0.4 μl aCSF alone or aCSFcontaining PcTX venom (500 ng/mL) 15 min before and following ischemia.In control mice with i.p. injection of saline and i.c.v. injection ofaCSF, 60 min MCAO induced an infarct volume of 123.6±5.3 mm³ (n=5, FIG.10C). In mice with i.p. injection of memantine and i.c.v. injection ofaCSF, the same duration of ischemia induced an infarct volume of73.8±6.9 mm³ (n=5, p<0.01). However, in mice injected with memantine andPcTX venom, an infarct volume of only 47.0±1.1 mm³ was induced (n=5,p<0.01 compared with both control and memantine groups, FIG. 10C). Thesedata suggest that blockade of homomeric ASIC1a may provide additionalprotection in in vivo ischemia in the setting of NMDA receptor blockade.Additional protection was also observed in ASIC1^(−/−) mice treated withpharmacologic NMDA blockade (FIG. 10D). In ASIC^(+/+) mice i.p. injectedwith saline or 10 mg/kg memantine, 60 min MCAO induced an infarct volumeof 101.4±9.4 mm³ or 61.6±12.7 mm³, respectively (n=5 in each group, FIG.10D). However, in ASIC1^(−/−) mice injected with memantine, the sameischemia duration induced an infarct volume of 27.7±1.6 mm³ (n=5),significantly smaller than the infarct volume in ASIC1^(+/+) miceinjected with memantine (p<0.05).

D. Discussion

Despite enormous recent progress defining cellular and molecularresponses of the brain to ischemia, there is no effective treatment forstroke patients. Most notable are the failures of multicenter clinicaltrials of glutamate antagonists (Lee et al. 1999 and Wahlgren and Ahmed2004). Here we demonstrate a new mechanism of ischemic brain injury andthe role of ischemic acidosis in this biology. We show that ischemicinjury in the setting of acidosis may occur via activation ofCa²⁺-permeable ASICs, a newly described class of ligand-gated channels(Waldmann et al., 1997a, and Waldmann and Lazdunski, 1998). This Ca²⁺toxicity may be independent of glutamate receptors or voltage-gated Ca²⁺channels.

Using whole-cell patch-clamp recording in mixed cortical cultures, wedemonstrate activation of ASIC currents in the range of pH_(e)(extracellular pH) that occurs commonly in ischemia. With Fura-2fluorescent imaging and ion substitution protocols, we show ASICs mayflux Ca²⁺ in cortical neurons and may do so in the presence of NMDA,AMPA, and voltage-gated Ca²⁺ channel blockade. Using in vitro celltoxicity models, we demonstrate that acidosis may induceglutamate-independent neuronal injury, which may be reduced by bothnonspecific and specific ASIC1a antagonists, and by lowering [Ca²⁺]_(e).In addition, we show that neurons and COS-7 cells lacking ASIC1a may beresistant to acid injury, while transfection of COS-7 cells withCa²⁺-permeable ASIC1a may result in acid sensitivity. Using in vivofocal ischemia models, we demonstrate that pharmacologic blockade ofASIC1a channels and ASIC1a gene knockout may both protect the brain fromischemic injury and may do so in the presence of NMDA blockade.

Local [H⁺] may be the agonist for ASICs functioning during normalsynaptic transmission in the brain (Wemmie et al., 2002). This signalingmay not be injurious. However, ASICs also may respond to the global,marked pH declines that may be occurring in the ischemic brain. Within Imin of global ischemia, pH_(e) falls from 7.2 to 6.5 (Simon et al.,1985), a level that may be sufficient to activate ASIC1a channels, whichhave a pH_(0.5) at 6.2. Remarkably, ischemia itself, modeled in vitro,markedly may enhance the magnitude of ASIC response at a given level ofacidosis, thus potentiating toxic Ca²⁺ loading in ischemic neurons.Furthermore, ischemia dramatically may reduce desensitization of ASICcurrents, signifying a possibility of long-lasting activity of ASICsduring prolonged ischemic acidosis in vivo.

It has been shown in intact animals that brief global reductions ofbrain pH to 6.5 alone do not produce brain injury (Litt et al., 1985),nor does hypoxia alone (Miyamoto and Auer, 2000, and Pearigen et al.,1996). However, our in vitro data suggest that the combination ofischemia (hypoxia) with acidosis (ischemic acidosis), as may occur invivo, may cause marked brain injury through ischemia enhancing the toxiceffect of ASIC1a channels. This argument is strongly supported by thefinding that both ASIC1a blockade and ASIC1a gene knockout producesubstantial (˜60%) reduction in infarct volume.

Acidosis, apart from affecting ischemic brain injury via ASICs, mayaffect the function of other channels as well. Particularly pertinent inischemia may be the acid blockade of the NMDA channels (Tang et al. 1990and Traynelis and Cull-Candy 1990), which may be protective against invitro ischemic neuronal injury (Kaku et al. 1993 and Giffard et al.1990). This NMDA blockade in the ischemic brain by acidosis might inpart explain the failure of NMDA antagonists in human stroke trials.Treatment of stroke with ASIC1a blockade may be particularly effective,as ischemic acidosis may be serving as an additional therapy by blockingNMDA function.

As our in vitro studies showing a protective effect of ASIC1a blockadewere performed in the presence of antagonists of NMDA, AMPA, andvoltage-gated Ca²⁺ channels, the findings reported here may offer a newand robust neuroprotective strategy for stroke, either alone or incombination with other therapies (MacGregor et al., 2003). Further, wedemonstrate in vivo that pharmacologic ASIC1a blockade or ASIC1a genedeletion may offer more potent neuroprotection against stroke than NMDAantagonism.

Together, our studies suggest that activation of Ca²⁺-permeable ASIC1amay be a novel, glutamate-independent biological mechanism underlyingischemic brain injury. As the regulation of other potentially protectiveASIC subunits also occurs in the ischemic brain (Johnson et al., 2001),these findings may help the design of novel therapeutic neuroprotectivestrategies for brain ischemia.

E. Experimental Procedures

1. Neuronal Culture

Following anesthesia with halothane, cerebral cortices were dissectedfrom E16 Swiss mice or P1 ASIC1^(+/+) and ASIC1^(−/−) mice and incubatedwith 0.05% trypsin-EDTA for 10 min at 37° C. Tissues were thentriturated with fire-polished glass pipettes and plated onpoly-L-ornithine-coated 24-well plates or 25×25 mm glass coverslips at adensity of 2.5×10⁵ cells per well or 10⁶ cells per coverslip. Neuronswere cultured with MEM supplemented with 10% horse serum (for E16cultures) or Neurobasal medium supplemented with B27 (for P1 cultures)and used for electrophysiology and toxicity studies after 12 days. Glialgrowth was suppressed by addition of 5-fluoro-2-deoxyuridine anduridine, yielding cultured cells with 90% neurons as determined by NeuNand GFAP staining (data not shown).

2. Electrophysiology

ASIC currents were recorded with whole-cell patch-clamp andfast-perfusion techniques. The normal extracellular solution (ECF)contained (in mM) 140 NaCl, 5.4 KCl, 25 HEPES, 20 glucose, 1.3 CaCl₂,1.0 MgCl₂, 0.0005 TTX (pH 7.4), 320-335 mOsm. For low pH solutions,various amounts of HCl were added. For solutions with pH<6.0, MESinstead of HEPES was used for more reliable pH buffering. Patchelectrodes contained (in mM) 140 CsF, 2.0 MgCl₂, 1.0 CaCl₂, 10 HEPES, 11EGTA, 4 MgATP (pH 7.3), 300 mOsm. The Na⁺-free solution consisted of 10mM CaCl₂, 25 mM HEPES with equiosmotic NMDG or sucrose substituting forNaCl (Chu et al., 2002). A multibarrel perfusion system (SF-77B, WarnerInstrument Co.) was employed for rapid exchange of solutions.

3. Cell Injury Assay—LDH Measurement

Cells were washed three times with ECF and randomly divided intotreatment groups. MK801 (10 μM), CNQX (20 μM), and nimodipine (5 μM)were added in all groups to eliminate potential secondary activation ofglutamate receptors and voltage-gated Ca²⁺ channels. Following acidincubation, neurons were washed and incubated in Neurobasal medium at37° C. LDH release was measured in culture medium using the LDH assaykit (Roche Molecular Biochemicals). Medium (100 μL) was transferred fromculture wells to 96-well plates and mixed with 100 μL reaction solutionprovided by the kit. Optical density was measured at 492 nm 30 minlater, utilizing a microplate reader (Spectra Max Plus, MolecularDevices). Background absorbance at 620 was subtracted. The maximalreleasable LDH was obtained in each well by 15 min incubation with 1%Triton X-100 at the end of each experiment.

4. Ca²⁺Imaging

Cortical neurons grown on 25×25 mm glass coverslips were washed threetimes with ECF and incubated with 5 μM fura-2-acetoxymethyl ester for 40min at 22° C., washed three times, and incubated in normal ECF for 30min. Coverslips were transferred to a perfusion chamber on an invertedmicroscope (Nikon TE300). Cells were illuminated using a xenon lamp andobserved with a 40×UV fluor oil-immersion objective lens, and videoimages were obtained using a cooled CCD camera (Sensys KAF 1401,Photometrics). Digitized images were acquired and analyzed in a PCcontrolled by Axon Imaging Workbench software (Axon Instruments). Theshutter and filter wheel (Lambda 10-2) were controlled by the softwareto allow timed illumination of cells at 340 or 380 nm excitationwavelengths. Fura-2 fluorescence was detected at emission wavelength of510 nm. Ratio images (340/380) were analyzed by averaging pixel ratiovalues in circumscribed regions of cells in the field of view. Thevalues were exported to SigmaPlot for further analysis.

5. Fluorescein-Diacetate Staining and Propidium Iodide Uptake

Cells were incubated in ECF containing fluorescein-diacetate (FDA) (5μM) and propidium iodide (PI) (2 μM) for 30 min followed by wash withdye-free ECF. Alive (FDA-positive) and dead (PI-positive) cells wereviewed and counted on a microscope (Zeiss) equipped with epifluorescenceat 580/630 nm excitation/emission for PI and 500/550 nm for FDA. Imageswere collected using an Optronics DEI-730 camera equipped with a BQ 8000sVGA frame grabber and analyzed using computer software (Bioquant, TN).

6. Transfection of COS-7 Cells

COS-7 cells were cultured in MEM with 10% HS and 1% PenStrep (GIBCO). At˜50% confluence, cells were cotransfected with cDNAs for ASICs and GFPin pc^(DNA3) vector using FuGENE6 transfection reagents (Roche MolecularBiochemicals). DNA for ASICs (0.75 μg) and 0.25 μg of DNA for GFP wereused for each 35 mm dish. GFP-positive cells were selected forpatch-clamp recording 48 hr after transfection. For stable transfectionof ASIC1a, 500 μg/mL G418 was added to culture medium I week followingthe transfection. The surviving G418-resistant cells were further platedand passed for >5 passages in the presence of G418. Cells were thenchecked with patch-clamp and immunofluorescent staining for theexpression of ASIC1a.

7. Oxygen-Glucose Deprivation

Neurons were washed three times and incubated with glucose-free ECF atpH 7.4 or 6.0 in an anaerobic chamber (Model 1025, Forma Scientific)with an atmosphere of 85% N₂, 10% H₂, and 5% CO₂ at 35° C.Oxygen-glucose deprivation (OGD) was terminated after 1 hr by replacingthe glucose-free ECF with Neurobasal medium and incubating the culturesin a normal cell culture incubator. With HEPES-buffered ECF used, 1 hrOGD slightly reduced pH from 7.38 to 7.28 (n=3) and from 6.0 to 5.96(n=4).

8. Focal Ischemia

Transient focal ischemia was induced by suture occlusion of the middlecerebral artery (MCAO) in male rats (SD, 250-300 g) and mice (withcongenic C57B16 background, ˜25 g) anesthetized using 1.5% isoflurane,70% N₂O, and 28.5% O₂ with intubation and ventilation. Rectal andtemporalis muscle temperature was maintained at 37° C.±0.5° C. with athermostatically controlled heating pad and lamp. Cerebral blood flowwas monitored by transcranical LASER doppler. Animals with blood flownot reduced below 20% were excluded.

Animals were killed with chloral hydrate 24 hr after ischemia. Brainswere rapidly removed, sectioned coronally at 1 mm (mice) or 2 mm (rats)intervals, and stained by immersion in vital dye (2%)2,3,5-triphenyltetrazolium hydrochloride (TTC). Infarction area wascalculated by subtracting the normal area stained with TTC in theischemic hemisphere from the area of the nonischemic hemisphere. Infarctvolume was calculated by summing infarction areas of all sections andmultiplying by slice thickness. Rat intraventricular injection wasperformed by stereotaxic technique using a microsyringe pump withcannula inserted stereotactically at 0.8 mm posterior to bregma, 1.5 mmlateral to midline, and 3.8 mm ventral to the dura. All manipulationsand analyses were performed by individuals blinded to treatment groups.

REFERENCES

Aarts, M., Iihara, K., Wei, W. L., Xiong, Z. G., Arundine, M.,Cerwinski, W., MacDonald, J. F. and Tymianski, M., 2003. A key role forTRPM7 channels in anoxic neuronal death. Cell 115, pp. 863-877.

Akopian, A. N., Chen, C. C., Ding, Y., Cesare, P. and Wood, J. N., 2000.A new member of the acid-sensing ion channel family. Neuroreport 11, pp.2217-2222.

Allen, N. J. and Attwell, D., 2002. Modulation of ASIC channels in ratcerebellar Purkinje neurons by ischemia-related signals. J. Physiol.543, pp. 521-529.

Alvarez de la Rosa, D., Zhang, P., Shao, D., White, F. and Canessa, C.M., 2002. Functional implications of the localization and activity ofacid-sensitive channels in rat peripheral nervous system. Proc. Natl.Acad. Sci. USA 99, pp. 2326-2331.

Alvarez de la Rosa, D., Krueger, S. R., Kolar, A., Shao, D.,Fitzsimonds, R. M. and Canessa, C. M., 2003. Distribution, subcellularlocalization and ontogeny of ASIC1 in the mammalian central nervoussystem. J. Physiol. 546, pp. 77-87.

Bassilana, F., Champigny, G., Waldmann, R., De Weille, J. R., Heurteaux,C. and Lazdunski, M., 1997. The acid-sensitive ionic channel subunitASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gatedNa+ channel with novel properties. J. Biol. Chem. 272, pp. 28819-28822.

Bederson, J. B., Pitts, L. H., Germano, S. M., Nishimura, M. C., Davis,R. L. and Bartkowski, H. M., 1986. Evaluation of2,3,5-triphenyltetrazolium chloride as a stain for detection andquantification of experimental cerebral infarction in rats. Stroke 17,pp. 1304-1308.

Benos, D. J. and Stanton, B. A., 1999. Functional domains within thedegenerin/epithelial sodium channel (Deg/ENaC) superfamily of ionchannels. J. Physiol. 520, pp. 631-644.

Benson, C. J., Eckert, S. P. and McCleskey, E. W., 1999. Acid-evokedcurrents in cardiac sensory neurons: a possible mediator of myocardialischemic sensation. Circ. Res. 84, pp. 921-928.

Bevan, S. and Yeats, J., 1991. Protons activate a cation conductance ina sub-population of rat dorsal root ganglion neurones. J. Physiol. 433,pp. 145-161.

Bianchi, L. and Driscoll, M., 2002. Protons at the gate: DEG/ENaC ionchannels help us feel and remember. Neuron 34, pp. 337-340.

Chen, C. C., England, S., Akopian, A. N. and Wood, J. N., 1998. Asensory neuron-specific, proton-gated ion channel. Proc. Natl. Acad Sci.USA 95, pp. 10240-10245.

Chen, C. C., Zimmer, A., Sun, W. H., Hall, J., Brownstein, M. J. andZimmer, A., 2002. A role for ASIC3 in the modulation of high-intensitypain stimuli. Proc. Natl. Acad Sci. USA 99, pp. 8992-8997.

Choi, D. W., 1988. Calcium-mediated neurotoxicity: relationship tospecific channel types and role in ischemic damage. Trends Neurosci. 11,pp. 465-469 a.

Choi, D. W., 1988. Glutamate neurotoxicity and diseases of the nervoussystem. Neuron 1, pp. 623-634 b.

Choi, D. W., 1995. Calcium: still center-stage in hypoxic-ischemicneuronal death. Trends Neurosci. 18, pp. 58-60.

Chu, X. P., Miesch, J., Johnson, M., Root, L., Zhu, X. M., Chen, D.,Simon, R. P. and Xiong, Z. G., 2002. Proton-gated channels in PC12cells. J. Neurophysiol. 87, pp. 2555-2561.

Dingledine, R., Borges, K., Bowie, D. and Traynelis, S. F., 1999. Theglutamate receptor ion channels. Pharmacol. Rev. 51, pp. 7-61.

Escoubas, P., De Weille, J. R., Lecoq, A., Diochot, S., Waldmann, R.,Champigny, G., Moinier, D., Menez, A. and Lazdunski, M., 2000. Isolationof a tarantula toxin specific for a class of proton-gated Na+ channels.J. Biol. Chem. 275, pp. 25116-25121.

Giffard, R. G., Monyer, H., Christine, C. W. and Choi, D. W., 1990.Acidosis reduces NMDA receptor activation, glutamate neurotoxicity, andoxygen-glucose deprivation neuronal injury in cortical cultures. BrainRes. 506, pp. 339-342.

Goldberg, M. P. and Choi, D. W., 1993. Combined oxygen and glucosedeprivation in cortical cell culture: calcium-dependent andcalcium-independent mechanisms of neuronal injury. J. Neurosci. 13, pp.3510-3524.

Grunder, S., Geissler, H. S., Bassler, E. L. and Ruppersberg, J. P.,2000. A new member of acid-sensing ion channels from pituitary gland.Neuroreport 11, pp. 1607-1611.

Immke, D. C. and McCleskey, E. W., 2001. Lactate enhances theacid-sensing Na+ channel on ischemia-sensing neurons. Nat. Neurosci. 4,pp. 869-870.

Jia, Z., Agopyan, N., Miu, P., Xiong, Z., Henderson, J., Gerlai, R.,Tavema, F. A., Velumian, A., MacDonald, J., Carlen, P. et al., 1996.Enhanced LTP in mice deficient in the AMPA receptor GluR2. Neuron 17,pp. 945-956.

Johnson, M. B., Jin, K., Minami, M., Chen, D. and Simon, R. P., 2001.Global ischemia induces expression of acid-sensing ion channel 2a in ratbrain. J. Cereb. Blood Flow Metab. 21, pp. 734-740.

Kaku, D. A., Goldberg, M. P. and Choi, D. W., 1991. Antagonism ofnon-NMDA receptors augments the neuroprotective effect of NMDA receptorblockade in cortical cultures subjected to prolonged deprivation ofoxygen and glucose. Brain Res. 554, pp. 344-347.

Kaku, D. A., Giffard, R. G. and Choi, D. W., 1993. Neuroprotectiveeffects of glutamate antagonists and extracellular acidity. Science 260,pp. 1516-1518.

Koh, J. Y. and Choi, D. W., 1987. Quantitative determination ofglutamate mediated cortical neuronal injury in cell culture by lactatedehydrogenase efflux assay. J. Neurosci. Methods 20, pp. 83-90.

Krishtal, O., 2003. The ASICs: signaling molecules?. Modulators? TrendsNeurosci. 26, pp. 477-483.

Krishtal, O. A. and Pidoplichko, V. I., 1981. A receptor for protons inthe membrane of sensory neurons may participate in nociception.Neuroscience 6, pp. 2599-2601.

Lee, J. M., Zipfel, G. J. and Choi, D. W., 1999. The changing landscapeof ischaemic brain injury mechanisms. Nature Suppl. 399, pp. A7-14.

Lingueglia, E., De Weille, J. R., Bassilana, F., Heurteaux, C., Sakai,H., Waldmann, R. and Lazdunski, M., 1997. A modulatory subunit of acidsensing ion channels in brain and dorsal root ganglion cells. J. Biol.Chem. 272, pp. 29778-29783.

Litt, L., Gonzalez-Mendez, R., Severinghaus, J. W., Hamilton, W. K.,Shuleshko, J., Murphy-Boesch, J. and James, T. L., 1985. Cerebralintracellular changes during supercarbia: an in vivo 31P nuclearmagnetic resonance study in rats. J. Cereb. Blood Flow Metab. 5, pp.537-544.

Longa, E. Z., Weinstein, P. R., Carlson, S. and Cummins, R., 1989.Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke 20, pp. 84-91.

MacGregor, D. G., Avshalumov, M. V. and Rice, M. E., 2003. Brain edemainduced by in vitro ischemia: causal factors and neuroprotection. J.Neurochem. 85, pp. 1402-1411.

McDonald, J. W., Bhattacharyya, T., Sensi, S. L., Lobner, D., Ying, H.S., Canzoniero, L. M. and Choi, D. W., 1998. Extracellular aciditypotentiates AMPA receptor-mediated cortical neuronal death. J. Neurosci.18, pp. 6290-6299.

McLennan, H., 1983. Receptors for the excitatory amino acids in themammalian central nervous system. Prog. Neurobiol. 20, pp. 251-271.

Meldrum, B. S., 1995. Excitatory amino acid receptors and their role inepilepsy and cerebral ischemia. Ann. N Y Acad Sci. 757, pp. 492-505.

Miyamoto, O. and Auer, R. N., 2000. Hypoxia, hyperoxia, ischemia, andbrain necrosis. Neurology 54, pp. 362-371.

Nedergaard, M., Kraig, R. P., Tanabe, J. and Pulsinelli, W. A., 1991.Dynamics of interstitial and intracellular pH in evolving brain infarct.Am. J. Physiol. 260, pp. R581-R588.

Pearigen, P., Gwinn, R. and Simon, R. P., 1996. The effects in vivo ofhypoxia on brain injury. Brain Res. 725, pp. 184-191.

Price, M. P., Snyder, P. M. and Welsh, M. J., 1996. Cloning andexpression of a novel human brain Na+ channel. J. Biol. Chem. 271, pp.7879-7882.

Price, M. P., Lewin, G. R., Mcllwrath, S. L., Cheng, C., Xie, J.,Heppenstall, P. A., Stucky, C. L., Mannsfeldt, A. G., Brennan, T. J.,Drummond, H. A. et al., 2000. The mammalian sodium channel BNC1 isrequired for normal touch sensation. Nature 407, pp. 1007-1011.

Price, M. P., Mcllwrath, S. L., Xie, J., Cheng, C., Qiao, J., Tarr, D.E., Sluka, K. A., Brennan, T. J., Lewin, G. R. and Welsh, M. J., 2001.The DRASIC cation channel contributes to the detection of cutaneoustouch and acid stimuli in mice. Neuron 32, pp. 1071-1083.

Rehncrona, S., 1985. Brain acidosis. Ann. Emerg. Med 14, pp. 770-776.

Rothman, S. M. and Olney, J. W., 1986. Glutamate and the pathophysiologyof hypoxic-ischemic brain damage. Ann. Neurol. 19, pp. 105-111.

Sattler, R., Xiong, Z., Lu, W. Y., Hafner, M., MacDonald, J. F. andTymianski, M., 1999. Specific coupling of NMDA receptor activation tonitric oxide neurotoxicity by PSD-95 protein. Science 284, pp.1845-1848.

Siesjo, B. K., Katsura, K. and Kristian, T., 1996. Acidosis-relateddamage. Adv. Neurol. 71, pp. 209-233.

Simon, R. P., Swan, J. H., Griffiths, T. and Meldrum, B. S., 1984.Blockade of N-methyl-D-aspartate receptors may protect against ischemicdamage in the brain. Science 226, pp. 850-852.

Simon, R. P., Benowitz, N., Hedlund, R. and Copeland, J., 1985.Influence of the blood-brain pH gradient on brain phenobarbital uptakeduring status epilepticus. J. Pharmacol. Exp. Ther. 234, pp. 830-835.

Sluka, K. A., Price, M. P., Breese, N. M., Stucky, C. L., Wemmie, J. A.and Welsh, M. J., 2003. Chronic hyperalgesia induced by repeated acidinjections in muscle is abolished by the loss of ASIC3, but not ASIC1.Pain 106, pp. 229-239.

Stenzel-Poore, M. P., Stevens, S. L., Xiong, Z., Lessov, N. S.,Harrington, C. A., Mori, M., Meller, R., Rosenzweig, H. L., Tobar, E.,Shaw, T. E. et al., 2003. Effect of ischaemic preconditioning on genomicresponse to cerebral ischaemia: similarity to neuroprotective strategiesin hibernation and hypoxia-tolerant states. Lancet 362, pp. 1028-1037.

Swanson, R. A., Farrell, K. and Simon, R. P., 1995. Acidosis causesfailure of astrocyte glutamate uptake during hypoxia. J. Cereb. BloodFlow Metab. 15, pp. 417-424.

Tang, C. M., Dichter, M. and Morad, M., 1990. Modulation of theN-methyl-D-aspartate channel by extracellular H+. Proc. Natl. Acad. Sci.USA 87, pp. 6445-6449.

Tariot, P. N., Farlow, M. R., Grossberg, G. T., Graham, S. M., McDonald,S. and Gergel, I., 2004. Memantine treatment in patients with moderateto severe Alzheimer disease already receiving donepezil: a randomizedcontrolled trial. JAMA 291, pp. 317-324.

Tombaugh, G. C. and Sapolsky, R. M., 1993. Evolving concepts about therole of acidosis in ischemic neuropathology. J. Neurochem. 61, pp.793-803.

Traynelis, S. F. and Cull-Candy, S. G., 1990. Proton inhibition ofN-methyl-D-aspartate receptors in cerebellar neurons. Nature 345, pp.347-350.

Ugawa, S., Ueda, T., Ishida, Y., Nishigaki, M., Shibata, Y. and Shimada,S., 2002. Amiloride-blockable acid-sensing ion channels are leading acidsensors expressed in human nociceptors. J. Clin. Invest. 110, pp.1185-1190.

Varming, T., 1999. Proton-gated ion channels in cultured mouse corticalneurons. Neuropharmacology 38, pp. 1875-1881.

Wahlgren, N. G. and Ahmed, N., 2004. Neuroprotection in cerebralischaemia: facts and fancies—the need for new approaches. Cerebrovasc.Dis. Supp. 17, pp. 153-166.

Waldmann, R. and Lazdunski, M., 1998. H(+)-gated cation channels:neuronal acid sensors in the NaC/DEG family of ion channels. Curr. Opin.Neurobiol. 8, pp. 418-424.

Waldmann, R., Champigny, G., Bassilana, F., Heurteaux, C. and Lazdunski,M., 1997a. A proton-gated cation channel involved in acid-sensing.Nature 386, pp. 173-177.

Waldmann, R., Bassilana, F., de Weille, J., Champigny, G., Heurteaux, C.and Lazdunski, M., 1997b. Molecular cloning of a non-inactivatingproton-gated Na+ channel specific for sensory neurons. J. Biol. Chem.272, pp. 20975-20978.

Waldmann, R., Champigny, G., Lingueglia, E., De Weille, J., Heurteaux,C. and Lazdunski, M., 1999. H(+)-gated cation channels. Ann. N Y Acad.Sci. 868, pp. 67-76.

Wemmie, J. A., Chen, J., Askwith, C. C., Hruska-Hageman, A. M., Price,M. P., Nolan, B. C., Yoder, P. G., Lamani, E., Hoshi, T., Freeman, J. H.and Welsh, M. J., 2002. The acid-activated ion channel ASIC contributesto synaptic plasticity, learning, and memory. Neuron 34, pp. 463-477.

Wemmie, J. A., Askwith, C. C., Lamani, E., Cassell, M. D., Freeman Jr.,J. H. and Welsh, M. J., 2003. Acid-sensing ion channel 1 is localized inbrain regions with high synaptic density and contributes to fearconditioning. J. Neurosci. 23, pp. 5496-5502.

Westergaard, E., 1969. The cerebral ventricles of the rat during growth.Acta Anat. (Basel) 74, pp. 405-423.

Xiong, Z., Lu, W. and MacDonald, J. F., 1997. Extracellular calciumsensed by a novel cation channel in hippocampal neurons. Proc. Natl.Acad. Sci. USA 94, pp. 7012-7017.

Yermolaieva, O., Leonard, A. S., Schnizler, M. K., Abboud, F. M. andWelsh, M. J., 2004. Extracellular acidosis increases neuronal cellcalcium by activating acid-sensing ion channel 1a. Proc. Natl. Acad.Sci. USA 101, pp. 6752-6757.

Ying, W., Han, S. K., Miller, J. W. and Swanson, R. A., 1999. Acidosispotentiates oxidative neuronal death by multiple mechanisms. J.Neurochem. 73, pp. 1549-1556.

Example 2 Time Window of PcTX Neuroprotection

This example describes exemplary experiments that measure theneuroprotective effect of PcTX venom at different times after onset ofstroke in rodents; see FIG. 11. Brain ischemia (stroke) was induced inrodents by mid-cerebral artery occlusion (MCAO). At the indicated timesafter induction, artificial cerebrospinal fluid (aCSF), PcTX venom (0.5μL, 500 ng/mL total protein), or inactivated (boiled) venom was infusedinto the lateral ventricles of each rodent. Administration of PcTX venomprovided a 60% reduction in stroke volume both at one hour and at threehours after stroke onset. Furthermore, substantial stroke volumereduction still may be maintained if treatment is withheld for fivehours after the onset of the MCAO. Accordingly, neuroprotection due toASIC inhibition may have an extended therapeutic time window afterstroke onset, allowing stroke subjects to benefit from treatmentperformed hours after the stroke began. This effect of ASIC blockade onstroke neuroprotection is far more robust than that of calcium channelblockade of the NMDA receptor (a major target for experimental stroketherapeutics) using a glutamate antagonist. No glutamate antagonist,thus far, has such a favorable profile as shown here forASIC1a-selective inhibition.

Example 3 Exemplary Cystine Knot Peptides

This example describes exemplary cystine knot peptides, includingfull-length PcTx1 and deletion derivatives of PcTx, which may bescreened in cultured cells, tested in ischemic animals (e.g., rodentssuch as mice or rats), and/or administered to ischemic human subjects;see FIGS. 12 and 13.

FIG. 12 shows the primary amino acid sequence (SEQ ID NO:1), inone-letter code, of an exemplary cystine knot peptide, PcTx1, indicatedat 50, with various exemplary peptide features shown relative to aminoacid positions 1-40. Peptide 50 may include six cysteine residues thatform cystine bonds 52, 54, 56 to create a cystine knot motif 58. Thepeptide also may include one or more beta sheet regions 60 and apositively charged region 62. An N-terminal region 64 and a C-terminalregion 66 may flank the cystine knot motif.

FIG. 13 shows a comparison of the PcTx1 peptide 50 of FIG. 12 alignedwith various exemplary deletion derivatives of the peptide. Thesederivatives may include an N-terminal deletion 70 (SEQ ID NO:2), apartial C-terminal deletion 72 (SEQ ID NO:3), a full C-terminal deletion74 (SEQ ID NO:4), and an N/C terminal deletion 76 (SEQ ID NO:5). Otherderivatives of PcTx1 may include any deletion, insertion, orsubstitution of one or more amino acids, for example, while maintainingsequence similarity or identity of at least about 25% or about 50% withthe original PcTx1 sequence.

Each PcTx1 derivative may be tested for its ability to inhibit ASICproteins selectively and/or for an effect, if any, on ischemia. Anysuitable test system(s) may be used to perform this testing includingany of the cell-based assay systems and/or animal model systemsdescribed elsewhere in the present teachings. The PcTx1 derivative alsoor alternatively may be tested in ischemic human subjects.

Example 4 Selectivity of PcTX Venom for ASIC1a

This example describes experiments that measure the selectivity of PcTXvenom (and thus PcTx1 toxin) for ASIC1a alone, relative to other ASICproteins or combinations of ASIC proteins expressed in cultured cells;see FIG. 14. COS-7 cells expressing the indicated ASIC proteins weretreated with PcTX venom (25 ng/mL on ASIC1a expressing cells and 500ng/mL on ASIC2a, ASIC3 or ASIC1a+2a expressing cells). Channel currentswere measured at the pH of half maximal channel activation (pH 0.5).PcTX venom largely blocked the currents mediated by ASIC1a homomericchannels at a protein concentration of 25 ng/mL, with no effect on thecurrents mediated by homomeric ASIC2a, ASIC3, or heteromericASIC1a/ASIC2a at 500 ng/mL (n=3-6, FIG. 14). At 500 ng/mL, PcTX venomalso did not affect the currents mediated by other ligand-gated channels(e.g. NMDA and GABA receptor-gated channels) and voltage-gated channels(e.g. Na+, Ca2+, and K+ channels) (n=4-5). These experiments indicatethat PcTX venom and thus PcTx1 peptide is a specific blocker forhomomeric ASIC1a. Using this cell-based assay system, the potency andselectivity of ASIC inhibition may be measured for various syntheticpeptides or other candidate inhibitors (e.g., see Example 3).

Example 5 Nasal Administration of PcTX Venom is Neuroprotective

This example describes exemplary data indicating the efficacy of nasallyadministered PcTX venom for reducing ischemia-induced injury in ananimal model system of stroke; see FIG. 15. Cerebral ischemia wasinduced in male mice by mid-cerebral artery occlusion. One hour afterocclusion was initiated animals were treated as controls or were treatedwith PcTX venom (50 μL of 5 ng/mL (total protein) PcTx venom introducedintranasally). Nasal administration of PcTX venom resulted in a 55%reduction in ischemia-induced injury (ischemic damage), as defined byinfarct volume, relative to control treatment. Nasal administration maybe via a spray that is deposited substantially in the nasal passagesrather than inhaled into the lungs and/or may be via an aerosol that isat least partially inhaled into the lungs. In some examples, nasaladministration may have a number of advantages over other routes ofadministration, such as more efficient delivery to the brain and/oradaptability for self-administration by an ischemic subject.

Example 6 Selected Embodiments

This example describes selected embodiments of the present teachings,presented as a series of indexed paragraphs.

1. A method for the treatment of ischemia-induced injury, comprising:

-   -   administering a therapeutically effective amount of an inhibitor        of an acid sensing ion channel (ASIC) family member to a subject        in need thereof.

2. The method of paragraph 1, wherein the step of administering isperformed on a stroke patient.

3. The method of paragraph 1, wherein the step of administering isperformed based on a risk of the subject for a future ischemic episodeor based or due to chronic ischemia.

4. The method of paragraph 1, wherein the step of administering isperformed to treat injury induced by ischemic heart disease.

5. The method of paragraph 1, wherein the step of administering includesadministering a plurality of doses of the inhibitor to the subject atdifferent times.

6. The method of paragraph 1, wherein the step of administering isperformed by injection of the inhibitor.

7. The method of paragraph 1, wherein the step of administering isperformed by ingesting or breathing the inhibitor.

8. The method of paragraph 1, wherein the step of administering includesadministering an inhibitor of ASIC1 family members.

9. The method of paragraph 8, wherein the step of administering includesadministering an inhibitor that is selective for ASIC1 family membersrelative to other ASIC family members.

10. The method of paragraph 1, wherein the step of administeringincludes administering an inhibitor of ASIC1a.

11. The method of paragraph 10, wherein the step of administeringincludes administering an inhibitor that is selective for ASIC1arelative to other ASIC family members.

12. The method of paragraph 11, wherein the step of administeringincludes administering an inhibitor that is specific for ASIC1 arelative to other ASIC family members.

13. The method of paragraph 1, wherein the step of administeringincludes administering a peptide having a cystine knot motif.

14. The method of paragraph 13, wherein the step of administeringincludes administering PcTx1, a toxin peptide from a tarantula species.

15. A method of screening for drugs to treat ischemia-induced injury,comprising:

-   -   selecting an assay system for measuring interaction with ASIC1a;    -   testing a set of compounds for interaction with ASIC1a in the        assay system to identify at least one compound that shows        interaction;    -   administering the at least one compound, or a structural        relative thereof, to a subject with ischemia to test the        efficacy of the at least one compound or the structural relative        for treatment of ischemia-induced injury.

16. The method of paragraph 15, wherein the step of selecting an assaysystem includes selecting an assay system that measures ion fluxmediated by ASIC1a.

17. The method of paragraph 16, wherein the step of selecting an assaysystem includes selecting an assay system that measures flux of calciummediated by ASIC1a.

18. The method of paragraph 16, wherein the step of testing a set ofcompounds includes testing the compounds for inhibition of the ion flux.

19. The method of paragraph 15, wherein the step of testing a set ofcompounds includes a step of testing the set of compounds for selectiveor specific inhibition of ASIC1a relative to at least one other ASICfamily member.

20. A composition for treating ischemia-induced injury, comprising:

-   -   an ASIC1a inhibitor configured as a medicament for        administration to human subjects.

21. The composition of paragraph 20, wherein the ASIC1a inhibitor isselective or specific for ASIC1a relative to each other ASIC familymember.

22. The composition of paragraph 20, wherein the ASIC1a inhibitor is apeptide having a cystine knot motif.

23. The composition of paragraph 22, wherein the peptide is PcTx1, atoxin from a tarantula species.

The disclosure set forth above may encompass one or more distinctinventions, with independent utility. Each of these inventions has beendisclosed in its preferred form(s). These preferred forms, including thespecific embodiments thereof as disclosed and illustrated herein, arenot intended to be considered in a limiting sense, because numerousvariations are possible. The subject matter of the inventions includesall novel and nonobvious combinations and subcombinations of the variouselements, features, functions, and/or properties disclosed herein. Thefollowing claims particularly point out certain combinations andsubcombinations regarded as novel and nonobvious. Inventions embodied inother combinations and subcombinations of features, functions, elements,and/or properties may be claimed in applications claiming priority fromthis or a related application. Such claims, whether directed to adifferent invention or to the same invention, and whether broader,narrower, equal, or different in scope to the original claims, also areregarded as included within the subject matter of the inventions of thepresent disclosure.

1. A method of treating ischemia, comprising: administering a therapeutically effective amount of an acid sensing ion channel la (ASIC1a) inhibitor to an ischemic subject in order to reduce injury resulting from ischemia.
 2. The method of claim 1, wherein the step of administering includes a step of administering an ASIC1a inhibitor that inhibits ASIC1a selectively relative to one or more other acid sensing ion channel (ASIC) family members.
 3. The method of claim 1, wherein the step of administering includes a step of administering an inhibitor that is selective for ASICI a relative to every other member of the ASIC family.
 4. The method of claim 1, wherein the step of administering is performed on a subject that has suffered a stroke, in order to reduce injury resulting from the stroke.
 5. The method of claim 1, wherein the step of administering includes a step of administering the inhibitor nasally, intrathecally, and/or epidurally.
 6. The method of claim 1, wherein the step of administering includes a step of administering a peptide that includes a cystine knot.
 7. The method of claim 1, wherein the step of administering includes a step of administering a peptide that is identical to or a derivative of PcTx1 (SEQ ID NO:1).
 8. The method of claim 7, wherein the peptide is a derivative that differs from PcTx1 by at least one deletion, substitution, and/or addition of one or more amino acids.
 9. The method of claim 1, the ASIC1a inhibitor being a first inhibitor, further comprising a step of administering a second inhibitor to the subject, the second inhibitor being configured to selectively inhibit at least one other channel that is not a member of the acid sensing ion channel family.
 10. The method of claim 1, wherein the step of administering includes a step of administering an inhibitor configured to inhibit ASIC1a channel activity at a concentration of inhibitor that is at least about ten-fold less than for inhibition of another ASIC family member.
 11. The method of claim 1, wherein the ischemia is stroke, and wherein the step of administering is performed at least about two hours after the onset of stroke on the ischemic subject to reduce injury induced by the stroke.
 12. A method of treating ischemia, comprising: administering a therapeutically effective amount of a cystine knot peptide to an ischemic subject in order to reduce injury resulting from ischemia.
 13. The method of claim 12, wherein the cystine knot peptide has an amino acid sequence with at least about 25% similarity to PcTx1 (SEQ ID:1).
 14. The method of claim 12, wherein the step of administering includes a step of administering the cystine knot peptide to a subject that has suffered a stroke.
 15. A method of identifying drugs for treating ischemia, comprising: obtaining one or more acid sensing ion channel 1a (ASIC1a) inhibitors; and testing the one or more ASIC1a inhibitors for an effect, if any, on an ischemic subject.
 16. The method of claim 15, wherein the step of obtaining includes a step of obtaining ASIC1a inhibitors that selectively inhibit ASIC1a relative to one or more other ASIC family members.
 17. The method of claim 15, wherein the step of obtaining includes a step of screening a plurality of compounds for selective inhibition of ASIC1a.
 18. The method of claim 17, wherein the step of screening includes a step of contacting cultured cells with the plurality of compounds.
 19. The method of claim 17, wherein the step of screening includes a step of detecting Ca²⁺ flux into the cultured cells.
 20. The method of claim 19, wherein the step of detecting Ca²⁺ flux is performed electrophysiologically, with a Ca²⁺ sensitive dye, and/or with dye that is sensitive to membrane potential.
 21. The method of claim 17, wherein the step of screening includes a step of screening a plurality of different peptides.
 22. The method of claim 17, wherein the step of screening includes a step of measuring inhibition of ASIC1a relative to inhibition of one or more other ASIC family members.
 23. The method of claim 15, wherein the step of testing includes steps of (1) inducing ischemia in at least one animal, (2) administering the one or more inhibitors to the at least one animal, and (3) detecting injury to the animal, if any, resulting from the ischemia.
 24. The method of claim 23, wherein the step of inducing ischemia includes a step of altering blood flow through a cerebral artery of the animal.
 25. The method of claim 15, wherein the step of testing includes (1) a step of selecting a plurality of human subjects that have suffered a stroke, and (2) a step of administering the one or more ASIC1a inhibitors to the human subjects to allow measurement of an effect, if any, of the one or more ASIC1a inhibitors on injury resulting from the stroke.
 26. A composition for treatment of ischemia, comprising: an acid sensing ion channel la (ASIC1a) inhibitor disposed in a vehicle at a concentration that provides a therapeutically effective amount of the ASIC1a inhibitor for treatment of ischemia when administered to an ischemic subject.
 27. The composition of claim 26, wherein the ASIC1a inhibitor inhibits acid ASIC1a selectively relative to one or more other acid sensing ion channel (ASIC) family members.
 28. The composition of claim 26, wherein the ASIC1a inhibitor is a peptide including a cystine knot.
 29. The composition of claim 26, wherein the inhibitor is PcTx1 (SEQ ID NO:1) or a derivative of PcTx1.
 30. A method of manufacturing a medicament for treatment of ischemia, comprising: obtaining an acid sensing ion channel la (ASIC1a) inhibitor; and combining the ASIC1a inhibitor with a vehicle to produce a medicament having a therapeutically effective concentration of the inhibitor for administration to an ischemic subject for treatment of ischemia.
 31. The method of claim 30, wherein the step of obtaining includes a step of obtaining an ASIC1a inhibitor that inhibits ASIC1a selectively relative to one or more other acid sensing ion channel (ASIC) family members.
 32. The method of claim 30, wherein the step of obtaining includes a step of obtaining PcTx1 (SEQ ID NO: 1) or a derivative of PcTx1.
 33. The method of claim 30, wherein the step of obtaining includes a step of obtaining an inhibitor that selectively inhibits ASIC1a relative to every other member of the ASIC family.
 34. The use of an acid sensing ion channel la (ASIC1a) inhibitor for the manufacture of a medicament to treat ischemia.
 35. The use of claim 34, the ASIC1a inhibitor being configured to inhibit ASIC1a selectively relative to one or more other acid sensing ion channel (ASIC) family members.
 36. The use of claim 34, wherein the inhibitor is used for the manufacture of a medicament to treat stroke.
 37. A method of manufacturing a medicament for treating ischemia, comprising formulating an ASIC1a inhibitor into such a medicament.
 38. The method of claim 37, wherein the ASIC1a inhibitor inhibits ASIC1a selectively relative to one or more other acid sensing ion channel (ASIC) family members. 